Over 15 million women suffer from urinary tract infections (UTI) annually in the U.S. and approximately 20-40% of these patients suffer from multiple recurrences. UTI is unique in its high prevalence of chronic/recurrent infections, which has caused a tremendous need for long-term conventional prophylactic therapy. This is leading to increased antimicrobial resistance of uropathogenic E. coli (UPEC) to first-line empiric therapy such as trimethoprim-sulfamethoxazole (TMP-SMZ). Over the past two decades, elucidation of bacterial pathogenic pathways in UPEC has revealed that the adhesin of type 1 pili, FimH, is an essential virulence factor and thus a novel therapeutic target for the prevention and treatment of UTI. This proposal uses an interdisciplinary approach blending medicinal chemistry, microbiology, and pharmacology to optimize potent, orally available small molecules called mannosides that attenuate acute virulence and recurrent infections by blocking the ability of FimH to bind to mannosylated receptors on bladder epithelial cells. This prevents the adherence to and invasion of UPEC into the bladder epithelium and ultimately prevents the formation of biofilm-like intracellular bacterial communities (IBCs). Further, this prevention of intracellular replication has the potential to mitigate the severity and frequency of recurrence. Rational structure-based ligand design of -D-mannose derivatives has led to the discovery of mannosides with tight FimH binding affinity and can both treat and prevent infection as measured in a murine model of UTI. This proposal is focused on further optimization of mannosides to identify preclinical leads therapeutics. Using knowledge of the structure activity relationships of current mannosides we have elucidated, improved mannosides will be synthesized and assessed in vitro for FimH inhibition and drug-like properties. The in vivo pharmacokinetics of lead mannoside FimH inhibitors will be evaluated in rats and then prioritized compounds in dogs. The most promising compounds will be tested for efficacy in preclinical pharmacodynamic mouse models of UTI. Mice are a relevant model for human UTI and will allow evaluation of in vivo efficacy for prevention/treatment of acute and chronic UTI. Current mannosides generate synergy with TMP-SMZ by excluding UPEC from the bladder epithelium, thus exposing the bacteria to concentrations of TMP-SMZ in the urine sufficient to kill an otherwise clinically resistant strain. The ability of mannoside/TMP-SMZ synergy to treat and prevent chronic/recurrent UTI, and generality to other antimicrobial-resistant strains using optimized inhibitors will be investigated. This proposal is designed to identify innovative, orally bioavailable small molecule therapeutics for the prevention and treatment of UTI. These drugs are also predicted to potentiate the activity of standard antimicrobials for the treatment of UTIs caused by resistant bacteria. Further, results of this proposal promise to serve as a pioneering model and precedent for developing new non-cytotoxic strategies for combating the rise in a myriad of other antibiotic resistant infections.